Detection of analytes present in exosomes

ABSTRACT

The invention provides methods for detection in stored biological samples of PrPsc and other analytes that are present in exosomes.

BACKGROUND OF THE INVENTION

PrPsc can be detected in buffy coat fractions of blood. Detection ofPrPsc from buffy coat fractions provides a way to concentrate the sampleand to remove interfering substances in plasma. However, the magnitudeof detection is inversely related to the age of the blood sample at timeof processing. That is, “aged” buffy coat samples retain less signalthan matched buffy coat samples that are processed immediately.Therefore, sensitive and specific methods of detection of PrPsc instored biological samples are needed in the art.

SUMMARY OF THE INVENTION

One embodiment of the invention provides methods for preparing abiological sample comprising leukocytes for detection of analytesassociated with exosomes. The methods comprise collecting a biologicalsample having leukocytes from a subject and adding an inhibitor ofN-SMase to the sample. The inhibitor of N-SMase can be glutathione(reduced form), spiroepoxide, N,N′-Bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-3,3′-p-phenylene-bis-acrylamidedihydrochloride, or ethylene glycol tetraacetic acid (EGTA). Theinhibitor can be added at a concentration of about 5 to about 75 mM. Theinhibitor of N-SMase can be added to the sample up to 1 hour aftercollection of the sample. The subject can be a human, cervid, caprine,bovine, ovine, rodent, mink, feline or a primate. The sample comprisingleukocytes can be a whole blood sample, a blood fraction containingleukocytes, a blood fraction containing peripheral blood mononuclearcells, tissue, or body fluids containing cellular fractions. The analytecan be PrPsc, a virus, or an exosome marker. The biological sample canbe purified or fractionated before or after the addition of theinhibitor of N-SMase to the sample.

Another embodiment of the invention provides methods of detection of ananalyte associated with exosomes. The methods comprise collecting abiological sample comprising leukocytes from a subject; adding aninhibitor of N-SMase to the sample; optionally storing the sample for aperiod of time; assaying the sample for the presence of the analyte. Theinhibitor of N-SMase can be glutathione (reduced form), spiroepoxide,N,N′-Bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-3,3′-p-phenylene-bis-acrylamidedihydrochloride, or ethylene glycol tetraacetic acid (EGTA). Theinhibitor can be added at a concentration of about 5 to about 75 mM. Thesubject can be a human, cervid, caprine, bovine, ovine, rodent, mink,feline or a primate. The sample comprising leukocytes can be a wholeblood sample, a blood fraction containing leukocytes, a blood fractioncontaining peripheral blood mononuclear cells, tissue, or body fluidscontaining cellular fractions. The analyte can be PrPsc, a virus, or anexosome marker. The biological sample can be purified or fractionatedbefore or after the addition of the inhibitor of N-SMase to the sample.

Therefore, the invention provides improved methods of detection ofanalytes associated with exosomes in samples comprising leukocytes,including samples that stored over time.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-B shows the effect of L-glutathione (reduced) and EGTA on thePrPsc signal obtained from the buffy coat of pre-clinical scrapie sheepwhen added to whole blood and the sample are stored for less than anhour or for 24 h.

FIG. 2A-B shows the effect of L-glutathione (reduced) and EGTA on thePrPsc signal obtained from the buffy coat of pre-clinical scrapie sheepwhen added to whole blood and the sample are stored for less than anhour or for 24 h.

FIG. 3A-B shows the effect of adding L-glutathione (reduced) or EGTA attime of collection to whole blood from normal and scrapie sheep andsubmitted to various storage conditions prior to processing.

FIG. 4A-B shows the evaluation of EGTA and either the oxidized orreduced form of L-glutathione at time of collection of whole blood fromnormal and scrapie sheep submitted to 24 h storage conditions at roomtemperature prior to processing, or processed immediately.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the singular forms “a,” “an”, and “the” include pluralreferents unless the context clearly dictates otherwise.

The detection of PrPsc in blood from antemortem transmissible spongiformencephalopathies (TSE) samples is problematic. The detection signal forPrPsc is lost partially or fully when PrPsc is present in plasma. Forexample, a full PrPsc detection signal (if any) cannot be obtained fromplasma samples supplemented with PrPsc from brain homogenates of scrapiesheep. However, when buffy coat fractions of blood samples are used todetect PrPsc, the magnitude of detection is inversely related to the ageof the blood sample at the time of detection. Therefore, samples must beassayed immediately after collection to obtain sensitive and specificresults.

PrPsc can be found in exosomes, which are membrane-bounded subcellularcompartments found in several cell types, including neurons and cells ofthe immune system that are involved in antigen presentation. Thesebilayer vesicles should not be confused with ribonuclease complexescapable of degrading RNAs, which can also be called exosomes. Neutralsphingomyelinase (N-SMase) activity is required for cells to releaseexosomes into their environment. It has now been discovered thattreatment of biological samples with inhibitors of N-SMase activityprevents the loss of PrPsc signal that occurs in the time intervalbetween sample collection and signal detection. For example,glutathione, a potent inhibitor of N-SMase, can preserve the detectionsignal in buffy coat fractions of blood samples from scrapie animalsthat were processed 24 hours post-collection. See Examples. Therefore,inhibitors of N-SMase can be added to the sample at or around the timeof sample collection to prevent shedding of analytes from cells viaexosomes, thereby preserving the ability to detect PrPsc or otheranalytes in samples after collection of the samples.

One embodiment of the invention provides methods for preparing orpreserving a biological sample for detection of analytes present in orassociated with exosomes or leukocytes. Analytes associated withexosomes or leukocytes are analytes that are present within exosomes orleukocytes or are part of the exosome or leukocytes (i.e., moleculesthat make up the exosome or leukocyte or are covalently ornon-covalently attached to the exosome or leukocyte). The methodscomprise collecting a biological sample comprising leukocytes from asubject and adding an inhibitor of N-SMase to the sample. The biologicalsample can be collected from any mammal, e.g., humans, cervids,caprines, bovines, ovines, rodents, mink, felines, or primates. Thebiological sample can be, e.g., a whole blood sample, a blood fractioncontaining leukocytes (e.g., a buffy coat fraction or a population ofperipheral blood mononuclear cells (which can be isolated using, e.g.,Ficoll-Hypaque techniques), tissue, and body fluids containing cellularfractions (e.g., cerebral spinal fluid, lymph fluid, whole blood, andurine). An inhibitor can be added to the biological sample itself, orcan be added to a purified portion or fraction of the sample. Forexample an inhibitor of N-SMase can be added to whole blood samples(which can then be fractionated or purified) or a buffy coat fraction ofthe whole blood sample.

The inhibitor of N-SMase can be, for example, L-glutathione (reducedform), spiroepoxide, N,N′-Bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-3,3′-p-phenylene-bis-acrylamidedihydrochloride (GW4869; Calbiochem, San Diego Calif.), ethylene glycoltetraacetic acid (EGTA), or combinations thereof; however, any inhibitorof N-SMase can be used in the methods of the invention. The inhibitorcan be added at a concentration of about 1, 2, 3, 4, 5, 10, 20, 30, 40,50, 60, 70, 75, 80, 90, 100, 125, 150 mM (or any range or value betweenabout 1 mM and 150 mM, e.g., about 5 mM to about 20 mM or about 5 mM toabout 75 mM). In one embodiment of the invention the inhibitor ofN-SMase is added to the biological sample immediately after collectionof the sample, but the inhibitor of N-SMase can be added to the sampleat about 2 hours, 1.5 hours, 1 hour, 50 minutes, 40 minutes, 30 minutes,20 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1minute (or any range or value between about 2 hours and 1 minute, e.g.about 1 hour to about 1 minute) or less after collection of the sample.The sample can be fractionated or purified before or after addition ofthe N-SMase inhibitor.

The analyte to be assayed can be, e.g., PrPsc, viruses that can be foundin leukocytes or exosomes (e.g., all major classes of exogenousvertebrate retroviruses, including the alpharetroviruses (e.g., avianmyeloblastosis virus and avian leukosis virus), betaretroviruses (e.g.,mouse mammary tumor virus), gammaretroviruses (e.g., murine leukemiavirus), deltaretroviruses (e.g., human T-lymphotrophic virus (HTLV-1)and bovine leukemia virus), lentiviruses (e.g., simian immunodeficiencyvirus, HIV, and Maedi-Visna virus), and spumaviruses), or any otheranalyte normally associated with an exosome or leukocyte, such as anendogenous exosome markers, e.g., annexins, acetylcholinesterase, CD24,flotillin, Rab, integrins, hsp70, Hsc73, Hsc90, subunits of trimeric Gproteins, Tsg101, tetraspanins (e.g., CD9, CD63, CD81, CD82, and CD151),and a variety of GPI (glycerol-phosphatidyl inositol)-anchored proteins,among others.

Infectious prions (PrPsc) are conformationally modified forms of thenormal cellular (PrPc) prion protein. The modification of PrPc to PrPsccan lead to the development of transmissible spongiform encephalopathies(TSEs), wherein PrPsc accumulates in the central nervous system andcauses neuropathologic changes and neurological dysfunction. TSEsinclude scrapie, which affects sheep and goats; bovine spongiformencephalopathy (BSE), which affects cattle; transmissible minkencephalopathy; feline spongiform encephalopathy; and chronic wastingdisease (CWD) of mule deer, white-tailed deer, black-tailed deer andelk. In humans, TSE diseases include kuru, Creutzfeldt-Jakob disease(CJD), Gerstmann-Straussler-Scheinker Syndrome (GSS), fatal insomnia andvariant Creutzfeldt-Jakob disease (vCJD).

One embodiment of the invention provides improved methods for detectionof an analyte associated with exosomes or leukocytes comprisingcollecting a biological sample comprising leukocytes from a subject,adding an inhibitor of N-SMase to the sample, optionally storing thesample for a period of time, and assaying the sample for the presence ofthe analyte. The sample can be stored for 5 minutes, 30 minutes, 1 hour,2 hours, 5 hours 10 hours, 24 hours, 48 hours, 72 hours, a week, orlonger. The sample can be stored at any temperature, e.g., −70, −20, 0,4, or 20 degrees Celsius or at room temperature.

Analytes can be detected using any immunological assay methods known inthe art including, e.g., enzyme immunoassays, western blot assays,competitive binding assays, radioimmunoassays, immunofluorescent assays,immuno-precipitation assays, chemiluminescent assays,immunohistochemical assays, dot blots, slot blots, ELISA assays,fluorescence-activated cell analyses, PCR assays, protein misfoldingcyclic amplification (PMCA) assays, quaking induced conversion (QUIC)assays, and nucleic acid detection assays. The assays can bequantitative or semi-quantitative.

For example, where PrPsc is an analyte, detection can be done using anantigen-capture enzyme immunoassay such as IDEXX HerdChek® BovineSpongiform Encephalopathy-Scrapie Antigen Test Kit. Where a virus is ananalyte, immunological or nucleic acid assays specific for that viruscan be used. Assays for endogenous exosome markers and analytesassociated with leukocytes are well known in the art.

All patents, patent applications, and other scientific or technicalwritings referred to anywhere herein are incorporated by reference intheir entirety. The invention illustratively described herein suitablycan be practiced in the absence of any element or elements, limitationor limitations that are not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising”,“consisting essentially of”, and “consisting of” may be replaced witheither of the other two terms, while retaining their ordinary meanings.The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention that in theuse of such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, but it is recognizedthat various modifications are possible within the scope of theinvention claimed. Thus, it should be understood that although thepresent invention has been specifically disclosed by embodiments,optional features, modification and variation of the concepts hereindisclosed may be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the description and the appended claims. Inaddition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

EXAMPLES

L-glutathione, an inhibitor of neutral sphingomyelinase (N-SMase), aswell as EGTA, a calcium chelator was used to preserve the PrPsc signalpresent in buffy coat preparations from scrapie blood. The L-glutathionewas added directly to the blood and stored under various conditions. ThePrPsc signal in buffy coat preparations was measured using the IDEXXHerdChek® Bovine Spongiform Encephalopathy-Scrapie Antigen Test Kit(“the IDEXX BSE/scrapie kit”).

L-glutathione (reduced) or EGTA was added at the time of collection towhole blood at 5 mM final concentration on the PrPsc signal of buffycoat isolated from whole blood from ARQ/ARQ scrapie sheep and submittedto various storage conditions prior to processing. FIG. 1 demonstratesthat L-glutathione (reduced) and EGTA preserve the PrPsc signal from thebuffy coat of pre-clinical scrapie sheep #1 (ARQ/ARQ) when added towhole blood and stored for 24 h. FIG. 1A shows the Optical Density (OD)signals from scrapie blood leukocytes (1×10⁷ cells/100μL) processedimmediately or after storage at 4° C. or room temperature (20-25° C.)for 24 hours. FIG. 1B shows tabular data of the same experiment showingpercentage change analysis. Percentage of samples processed after 24 hrepresents the residual signal relative to the signal of the sampleprocessed immediately, which is defined as 100% signal for eachtreatment condition. Bolded numbers indicate >10% decrease in signal,while underlined numbers indicate little or no significant change. ThePrPsc signal in buffy coat preparations was measured using the IDEXXBSE/scrapie kit.

A second trial to test the effect of adding L-glutathione (reduced) orEGTA at time of collection to whole blood at 5 mM final concentration onthe PrPsc signal of buffy coat isolated from whole blood from ARQ/ARQscrapie sheep and submitted to various storage conditions prior toprocessing is shown in FIG. 2. L-glutathione (reduced) and EGTA preservethe PrPsc signal from the buffy coat of pre-clinical scrapie sheep #2(ARQ/ARQ) when added to whole blood and stored for 24 h. FIG. 2A showsthe OD signals from scrapie blood leukocytes (1×10⁷ cells/100μL)processed immediately or after storage at 4° C. or room temperature(20-25° C.) for 24 hours. FIG. 2B shows tabular data of the sameexperiment showing percentage change analysis. Percentage of samplesprocessed after 24 h represents the residual signal relative to thesignal of the sample processed immediately, which is defined as 100%signal for each treatment condition. Bold numbers indicate >10% decreasein signal, while underlined numbers indicate little or no significantchange. The PrPsc signal in buffy coat preparations was measured usingthe IDEXX BSE/scrapie kit.

The effect of adding L-glutathione (reduced) or EGTA at time ofcollection to whole blood from normal and ARQ/ARQ scrapie sheep andsubmitted to various storage conditions prior to processing is shown inFIG. 3. FIG. 3 shows the effect of overnight incubation of whole bloodfrom scrapie (ARQ/ARQ) and normal sheep with L-glutathione (reduced).FIG. 3A shows OD signals from scrapie blood leukocytes (1×10⁷cells/100μL) processed immediately or after storage at 4° C. or roomtemperature (20-25° C.) for 24 hours. FIG. 3B shows OD signals fromnormal blood leukocytes (1×10⁷ cells/100μL) processed immediately orafter storage at 4° C. or room temperature (20-25° C.) for 24 hours.Tabular data of the same experiment showing percentage change analysis.Percentage of samples processed after 24 h represents the residualsignal relative to the signal of the sample processed immediately, whichis defined as 100% signal for each treatment condition. Bold numbersindicate >10% decrease in signal, while underlined numbers indicatelittle or no significant change. The PrPsc signal in buffy coatpreparations was measured using the IDEXX BSE/scrapie kit.

FIG. 4 shows the evaluation of EGTA and either the oxidized or reducedform of L-glutathione at time of collection of whole blood from normaland scrapie sheep (VRQ/VRQ) submitted to 24 h storage condition at roomtemperature prior to processing, or processed immediately. FIG. 4 showsthe effect of L-glutathione (reduced) and EGTA on blood from VRQ/VRQscrapie and normal sheep. FIG. 4A shows OD signals from scrapie andnormal blood leukocytes (1×10⁷ cells/100 μL) processed immediately orafter storage at room temperature (20-25° C.) for 24 hours. FIG. 4Bshows tabular data of the same experiment showing percentage loss ofsignal relative to the signal of the sample processed immediately, whichis defined as 100% signal for each treatment condition (also shown as 0%loss). Note the lack of effect of the oxidized L-glutathione compared tothe reduced form. For the oxidized glutathione, the percentage loss wascalculated relative to the untreated sample processed immediately.

Therefore, the reduced form of L-glutathione and EGTA allow for thepreservation of the PrPsc harbored in the buffy coat of blood fromscrapie sheep of both ARQ/ARQ and VRQ/VRQ genotype backgrounds afterthese additives were incorporated in the blood and stored for at least24 h.

We claim:
 1. A method for preparing a biological sample comprisingleukocytes for detection of analytes associated with exosomescomprising: (a) collecting a biological sample comprising leukocytesfrom a subject; (b) adding an inhibitor of N-SMase to the sample.
 2. Themethod of claim 1 wherein the inhibitor of N-SMase is a reduced form ofglutathione, spiroepoxide, N,N′-Bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-3,3′-p-phenylene-bis-acrylamide dihydrochloride,or ethylene glycol tetraacetic acid (EGTA).
 3. The method of claim 1,wherein the inhibitor is added at a concentration of about 5 to about 75mM.
 4. The method of claim 1, wherein the inhibitor of N-SMase is addedto the sample up to 1 hour after collection of the sample.
 5. The methodof claim 1, wherein the subject is a human, cervid, caprine, bovine,ovine, rodent, mink, feline or a primate.
 6. The method of claim 1,wherein the sample comprising leukocytes is a whole blood sample, ablood fraction containing leukocytes, a blood fraction containingperipheral blood mononuclear cells, tissue, or body fluids containingcellular fractions.
 7. The method of claim 1, wherein the analyte isPrPsc, a virus, or an exosome marker.
 8. The method of claim 1, whereinthe biological sample is purified or fractionated before or after theaddition of the inhibitor of N-SMase to the sample.
 9. A method ofdetection of an analyte associated with exosomes comprising (a)collecting a biological sample comprising leukocytes from a subject; (b)adding an inhibitor of N-SMase to the sample; (c) optionally storing thesample for a period of time; (d) assaying the sample for the presence ofthe analyte.
 10. The method of claim 9, wherein the inhibitor of N-SMaseis a reduced form of glutathione, spiroepoxide,N,N′-Bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-3,3′-p-phenylene-bis-acrylamidedihydrochloride, or ethylene glycol tetraacetic acid (EGTA).
 11. Themethod of claim 9, wherein the inhibitor is added at a concentration ofabout 5 to about 75 mM.
 12. The method of claim 9, wherein the subjectis a human, cervid, caprine, bovine, ovine, rodent, mink, feline or aprimate.
 13. The method of claim 9, wherein the sample comprisingleukocytes is a whole blood sample, a blood fraction containingleukocytes, a blood fraction containing peripheral blood mononuclearcells, tissue, or body fluids containing cellular fractions.
 14. Themethod of claim 9, wherein the analyte is PrPsc, a virus, or an exosomemarker.
 15. The method of claim 9, wherein the biological sample ispurified or fractionated before or after the addition of the inhibitorof N-SMase to the sample.